Biochemical and molecular characterization of fibrinolytic enzyme purified from Pleurotus ostreatus | ||||
| Arab Journal of Biotechnology | ||||
| Volume 19, Issue 1, June 2016, Page 17-34 PDF (1007.84 K) | ||||
| Document Type: Original Article | ||||
| DOI: 10.21608/arjb.2016.428487 | ||||
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| Abstract | ||||
| The crude fibrinolytic enzyme was purified to full homogeneity using fractional precipitation by ammonium sulphate, DEAE cellulose chromatography and gel filtration on Sephadex-G100. An over all of 69%-fold purification with 2.5 recovery was obtained. The apparent molecular mass of the purified enzyme was estimated to be 44 kDa using SDS-PAGE. The optimum pH and temperature of the purified enzyme were 6 and 35˚C, respectively. The fibrinolytic enzyme was completely inhibited by Hg2+ and partially inhibited by Cu2+, Ni2+ and Al3+. Its activity strongly enhanced by Zn2+, Mg2+, Ca2+, K+ and Fe2+ in descending order. EDTA and EGTA inhibited the enzyme activity suggesting that it is a metalloprotease. The enzyme was also inhibited by the serine inhibitors PMSF and aprotinin. The pure enzyme showed strong specificity to fibrin as substrate in vitro. It was also specific to gelatin and fibrinogen but not specific to casein, elastin and egg albumin. The amidolytic activity toward synthetic substrates showed high specificity to the synthetic peptide N-Succinyl-Ala-Ala-Pro-Phe-pNA suggesting that it is a chymotrypsin-like protease. The PoFR-cDNA encoding gene was cloned in E. coli (α-DH5) and its nucleotide sequence was determined (GenBank accession no. AB551656). The PoFR-cDNA was found to consist of 845 bp in an Open Reading Frame (ORF) encoding 281 amino acids. The sequence showed high degree of homology with the fibrinolytic enzyme gene from P. ostreatus fruiting bodies (AY640032.1). In vivo assay using thrombus induced mice showed that the enzyme exerted thrombolysis in the mice blood marked by decrease in hematocrit percentage and prolonged prothrombin time (PT) and thrombin time (TT). | ||||
| Keywords | ||||
| Pleurotus ostreatus; Fibrinolytic enzymes; Thrombosis; Thrombolysis | ||||
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